Sensitive and specific CRISPR diagnostics

Methods are needed that can easily detect nucleic acids that signal the presence of pathogens, even at very low levels. Gootenberg et al. combined the allele-specific sensing ability of CRISPR-Cas13a with recombinase polymerase amplification methods to detect specific RNA and DNA sequences. The method successfully detected attomolar levels of Zika virus, as well as the presence of pathogenic bacteria. It could also be used to perform human genotyping from cell-free DNA.

The ability to rapidly detect nucleic acids with high sensitivity and single-base specificity on a portable platform may aid in disease diagnosis and monitoring, epidemiology, and general laboratory tasks. Although methods exist for detecting nucleic acids (1–6), they have trade-offs among sensitivity, specificity, simplicity, cost, and speed. Microbial clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) adaptive immune systems contain programmable endonucleases that can be leveraged for CRISPR-based diagnostics (CRISPR-Dx). Although some Cas enzymes target DNA (7, 8), single-effector RNA-guided ribonucleases (RNases), such as Cas13a (previously known as C2c2) (8), can be reprogrammed with CRISPR RNAs (crRNAs) to provide a platform for specific RNA sensing (9–12). On recognition of its RNA target, activated Cas13a engages in “collateral” cleavage of nearby nontargeted RNAs (10). This crRNA-programmed collateral-cleavage activity allows Cas13a to detect the presence of a specific RNA in vivo by triggering programmed cell death (10) or in vitro by nonspecific degradation of labeled RNA (10, 12). Here we describe Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), an in vitro nucleic acid–detection platform with attomolar sensitivity based on nucleic acid amplification and Cas13a-mediated collateral cleavage of a reporter RNA (12), allowing for real-time detection of the target (Fig. 1A).

We first determined the sensitivity of SHERLOCK for detection of RNA (when coupled with reverse transcription) or DNA targets. We achieved single-molecule sensitivity for both RNA and DNA, as verified by digital-droplet polymerase chain reaction (ddPCR) (Fig. 1, C and D, and fig. S4, B and C). Attomolar sensitivity was maintained when we combined all SHERLOCK components in a single reaction, demonstrating the viability of this platform as a point-of-care diagnostic (fig. S4D). SHERLOCK has similar levels of sensitivity to those of ddPCR and quantitative PCR (qPCR), two established sensitive nucleic acid–detection approaches, whereas RPA alone was not sensitive enough to detect low levels of target (fig. S5, A to D). Moreover, SHERLOCK shows less variation than ddPCR, qPCR, and RPA, as measured by the coefficient of variation across replicates (fig. S5, E and F).

We next examined whether SHERLOCK would be effective in infectious disease applications that require high sensitivity. We produced lentiviruses harboring genome fragments of either Zika virus (ZIKV) or the related flavivirus dengue (DENV) (19) (Fig. 2A). SHERLOCK detected viral particles down to 2 aM and could discriminate between ZIKV and DENV (Fig. 2B). To explore the potential use of SHERLOCK in the field with paper spotting and lyophilization (1), we first demonstrated that Cas13a-crRNA complexes that were lyophilized and subsequently rehydrated (13) could detect 20 fM of nonamplified ssRNA 1 (fig. S6A) and that target detection was also possible on glass fiber paper (fig. S6B). The other components of SHERLOCK are also amenable to freeze-drying: RPA is provided as a lyophilized reagent at ambient temperature, and we previously demonstrated that T7 polymerase tolerates freeze-drying (2). In combination, freeze-drying and paper spotting the Cas13a detection reaction resulted in levels of sensitive detection of ssRNA 1 comparable to those of aqueous reactions (fig. S6, C to E). Although paper spotting and lyophilization slightly reduced the absolute signal of the readout, SHERLOCK (Fig. 2C) could readily detect mock ZIKV virus at concentrations as low as 20 aM (Fig. 2D).

SHERLOCK can also detect ZIKV in clinical isolates (serum or urine), where titers can be as low as 2 × 103 copies/ml (3.2 aM) (20). ZIKV RNA extracted from patient serum or urine samples and reverse transcribed into cDNA (Fig. 2E) could be detected at concentrations down to 1.25 × 103 copies/ml (2.1 aM), as verified by qPCR (Fig. 2F). Furthermore, the signal from patient samples was predictive of ZIKV RNA copy number and could be used to predict viral load (fig. S6F). To simulate sample detection without nucleic acid purification, we measured detection of ssRNA 1 spiked into human serum and found that Cas13a could detect RNA in reactions containing as much as 2% serum (fig. S6G).

Another important epidemiological application for CRISPR-Dx is the identification of bacterial pathogens and detection of specific bacterial genes. We targeted the V3 region of the 16S ribosomal RNA (rRNA) gene, where conserved flanking regions allow universal RPA primers to be used across bacterial species and the variable internal region allows for differentiation of species. In a panel of five possible targeting crRNAs for different pathogenic strains and genomic DNA (gDNA) isolated from Escherichia coli and Pseudomonas aeruginosa (Fig. 2G), SHERLOCK correctly genotyped strains and showed low cross-reactivity (Fig. 2H). Additionally, we were able to use SHERLOCK to distinguish between clinical isolates of Klebsiella pneumoniae with two different resistance genes: Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase 1 (NDM-1) (21) (fig. S7).

To increase the specificity of SHERLOCK, we introduced synthetic mismatches in the crRNA:target duplex that enable LwCas13a to discriminate between targets that differ by a single-base mismatch (fig. S8, A and B). We designed multiple crRNAs with synthetic mismatches in the spacer sequences to detect either the African or American strains of ZIKV (Fig. 3, A and B) and strain 1 or 3 of DENV (Fig. 3, C and D). Synthetic mismatch crRNAs detected their corresponding strains with significantly higher signal (two-tailed Student’s t test, P < 0.01) than the off-target strain, allowing for robust strain discrimination on the basis of single mismatches (Fig. 3, B to D, and fig. S8C). Further characterization revealed that Cas13a detection achieves maximal specificity while maintaining on-target sensitivity when a mutation is in position 3 of the spacer and the synthetic mismatch is in position 5 (figs. S9 and S10).

Fig. 3Cas13a detection can discriminate between similar viral strains.

(A) Schematic of ZIKV strain target regions and the crRNA sequences used for detection. SNPs in the target are highlighted red or blue, and synthetic mismatches in the guide sequence are in red. (B) Highly specific detection of strain SNPs allows for the differentiation of ZIKV African versus American RNA targets with Cas13a. (C) Schematic of DENV strain target regions and the crRNA sequences used for detection. SNPs in the target are highlighted red or blue, and synthetic mismatches in the guide sequence are in red. (D) Highly specific detection of strain SNPs allows for the differentiation of DENV strain 1 versus strain 3 RNA targets with Cas13a. (B and D) n = 2 technical replicates, two-tailed Student’s t test; *P < 0.05, **P < 0.01, and ***P < 0.001; bars represent mean ± SEM.

The ability to detect single-base differences opens the opportunity for using SHERLOCK for rapid human genotyping. We chose five loci spanning a range of health-related single-nucleotide polymorphisms (SNPs) (table S1) and benchmarked SHERLOCK detection using genotyping data from 23andMe, a genetic testing company, as the “gold standard” at these SNPs (22) (Fig. 4A). We collected saliva from four human subjects with diverse genotypes across the loci of interest and extracted gDNA through either column purification or direct heating for 5 min (13). SHERLOCK distinguished alleles with high significance and with enough specificity to infer both homozygous and heterozygous genotypes (Fig. 4B and figs. S11 and S12).

Finally, we sought to determine if SHERLOCK could detect low-frequency cancer mutations in cell-free DNA (cfDNA) fragments, which is challenging because of the high levels of wild-type DNA in patient blood (23–25). We first found that SHERLOCK could detect ssDNA 1 at attomolar concentrations diluted in a background of gDNA (fig. S13A). Next, we found that SHERLOCK was also able to detect SNP-containing alleles (fig. S13, B and C) at levels as low as 0.1% of background DNA, which is in the clinically relevant range. We then demonstrated that SHERLOCK could detect two different cancer mutations, EGFR L858R (L, Leu; R, Arg) and BRAF V600E (V, Val; E, Glu), in mock cfDNA samples with allelic fractions as low as 0.1% (Fig. 4, C to F) (13).

The SHERLOCK platform lends itself to further applications, including (i) general RNA and DNA quantitation in lieu of specific qPCR assays, such as TaqMan; (ii) rapid, multiplexed RNA-expression detection; and (iii) other sensitive detection applications, such as detection of nucleic acid contamination. Additionally, Cas13a could potentially detect transcripts within biological contexts and track allele-specific expression of transcripts or disease-associated mutations in live cells. We have shown that SHERLOCK is a versatile, robust method that can rapidly detect single molecules of DNA or RNA, suitable for applications involving infectious disease and sensitive genotyping. A SHERLOCK paper test can be redesigned and synthesized in a matter of days for as low as $0.61 per test (table S2) with confidence, as almost every crRNA tested resulted in high sensitivity and specificity. These qualities highlight the power of CRISPR-Dx and open new avenues for rapid, robust, and sensitive detection of biological molecules.

Acknowledgments: We thank F. Chen, V. Rusu, R. Gupta, D. Daniels, C. Garvie, I. Finkelstein, V. Adalsteinsson, A. Das, E. S. Lander, R. Macrae, and R. Belliveau for discussions and support. Human genotyping data were collected with the informed consent of the subjects and in consent with the guidelines of the approved Massachusetts Institute of Technology (MIT) institutional review board (IRB) protocol IRB-4062. O.O.A. is supported by a Paul and Daisy Soros Fellowship and a National Defense Science and Engineering Fellowship. J.S.G. is supported by a U.S. Department of Energy Computational Science Graduate Fellowship. R.P.B, J.L, and D.T.H. are supported by the NIH through a National Institute of Allergies and Infectious Diseases grant (R01AI117043). A.J.D. is supported by an NSF Graduate Research Fellowship and an Air Force Office of Scientific Research grant (FA9550-14-1-0060). Zika work was partially funded by M. and L. Benioff to P.C.S., and antibiotic resistance work was partially funded by J. and A. Bekenstein to D.T.H. A.R. is supported by the Howard Hughes Medical Institute. J.J.C. is supported by the Defense Threat Reduction Agency grant HDTRA1-14-1-0006, the Paul G. Allen Frontiers Group, and the Wyss Institute. F.Z. is a New York Stem Cell Foundation–Robertson Investigator. F.Z. is supported by the NIH through National Institute of Mental Health grants (5DP1-MH100706 and 1R01-MH110049); the NSF; the Howard Hughes Medical Institute; the New York Stem Cell, Simons, Paul G. Allen Family, and Vallee Foundations; and J. and P. Poitras, R. Metcalfe, and D. Cheng. A.R. is a member of the Scientific Advisory Board for ThermoFisher Scientific. J.S.G., O.O.A., R.P.B., A.R., E.V.K., D.T.H., P.C.S., J.J.C., and F.Z. have filed patent applications relating to the work in this manuscript, including J.S.G., O.O.A., E.V.K., and F.Z. on international application no. PCT/US2016/038258 filed 18 June 2015 (CRISPR-C2c2 systems and uses thereof); J.S.G., O.O.A., and F.Z. on U.S. provisional patent application no. 62/351,662 filed 17 June 2016 (CRISPR-C2c2 systems and diagnostic uses thereof); J.S.G., O.O.A., J.J.C., and F.Z. on U.S. provisional patent application no. 62/432,553 filed 9 December 2016 (SHERLOCK diagnostic); J.S.G., O.O.A., P.C.S., J.J.C., and F.Z. on U.S. provisional patent application no. 62/471,917 filed 15 March 2017 (viral application of SHERLOCK); J.S.G., O.O.A., A.R., J.J.C., and F.Z. on U.S. provisional patent application no. 62/471,931 filed 15 March 2017 (mutation detection with SHERLOCK); J.S.G., O.O.A., R.P.B., D.T.H., J.J.C., and F.Z. on U.S. provisional patent application no. 62/471,936 filed 15 March 2017 (bacterial applications of SHERLOCK); and J.S.G., O.O.A., J.J.C., and F.Z. on U.S. provisional patent application no. 62/471,940 filed 15 March 2017 (devices). Each patent application relates to CRISPR-C2c2 systems, specific uses, and improved uses thereof for diagnostic application filed by Broad, Harvard, Massachusetts General Hospital, MIT, and NIH. Cas13a/C2c2 expression plasmids are available from Addgene under a Uniform Biological Material Transfer Agreement.